鲆鲽鱼类变态时期相关基因的克隆与分析
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摘要
鲆鲽鱼类与其他硬骨鱼类最明显的区别是脊椎的不对称性:两个眼睛都位于身体的一侧(有眼侧)。这一脊椎的不对称性的变化发生在鲆鲽鱼类的变态时期。变态时,鲆鲽鱼类的一只眼睛从一侧偏转到另一侧,由脊椎对称的浮游性鱼类发育成脊椎不对称的底栖型鱼类,同时也发生了很多其他形态学和生理学变化。但是,导致鲆鲽鱼类变态及眼睛偏转方向的分子机制一直没有定论。
左偏的牙鲆与右偏石鲽的杂交子代基于相同的遗传背景且在变态时眼睛向不同方向偏转,成为了研究鲆鲽鱼类变态和眼睛偏转方向的难得的实验材料。本实验运用了差异显示技术(DDRT-PCR)和代表性差异杂交(RDA)两种基因筛选方法用来筛选变态前期的左偏和右偏杂交种中差异表达的基因,分别筛选出23和47个片段。实验结果显示RDA是基因筛选的可行方法之一。大部分的片段与眼睛偏转方向都没有直接关系,但是RDA筛选出的肌肉型肌酸激酶1(CK-M1)和胰蛋白酶原前体2(TR2P)经检验与鲆鲽鱼类的变态有关。
肌肉型肌酸激酶1作为催化能量产生的重要催化剂之一,从变态前与变态前期的牙鲆仔鱼的RDA实验中筛选出经克隆测序,得到全序列。其序列与其他鱼类和哺乳动物的CK-M1的相似性很高。在牙鲆成鱼中,CK-M1在各个组织中都有较高的表达,特别是肌肉,内脏和鳃中。整体原位杂交和实时定量PCR结果显示CK-M1在变态期间的牙鲆仔鱼中的表达情况与变态紧密相关,随着变态的开始增加到高峰,之后下降,变态结束后,其表达量也下降到与变态前一样的水平。这些可以说明,CK-M1与牙鲆仔鱼的变态过程有着密切的关系。
胰蛋白酶原前体2是动物消化道中由胰腺细胞分泌的重要的消化酶之一。从牙鲆与石鲽杂交种的RDA实验中筛选出经分别在牙鲆和石鲽中克隆测序,分别得到全序列。其序列与其他鱼类的TR2P的相似性较高。在牙鲆成鱼中,TR2P在各个组织中都有表达,在肠道中的表达量最高。实时定量PCR结果显示TR2P在变态期间的牙鲆仔鱼中的表达情况与变态紧密相关,随着变态的开始突增,之后稍有下降,但一直维持高于变态前仔鱼的表达量直到变态完毕。可以说明,TR2P与牙鲆仔鱼的变态过程有着密切的关系。而且,RDA右偏库中筛选出的TR2P片段与同是右偏的父本石鲽的TR2P之间相似性高于与母本牙鲆,猜测其与鲆鲽鱼类的眼睛偏转方向相关。
Vertebral asymmetry in flatfish is the most significant among vertebrates,with both eyes being located on only one side(the ocular side) of the body.Changes in body proportion occur during flatfish metamorphosis,which exhibit extensive morphological and functional changes in various organs.There is little molecular reason to help understand the mechanism of flounders' eye migration,but still need to be proved.
The hybrids between Japanese flounder and stone flounder showed mixed eye-location:with both dextral type and sinistral type,and thus become good samples for studying the eye-migration problems,mRNAs from 22dph sinistral and dextral hybrids larvae were screened with the classical differential display RT-PCR (DD-RT-PCR) and representational difference analysis of cDNA(cDNA-RDA),and 23 and 47 putative fragments were isolated respectively.Most of the fragments isolated were not involved into eye migration specifically.But cDNA fragments of creatine kinase and trypsinogen 2 precursor genes isolated by cDNA-RDA exhibited stage specific expression patterns,indicating that they were related with energy supply and physiological changes during metamorphosis of flatfish.The present results showed that cDNA-RDA is one of the capable methods in isolating differentially expressed genes.
Creatine kinase(CK) catalyzes the reversible reaction of phosphorylation of creatine by ATP,which produces phosphorycreatine,a high-energy phosphagen in all vertebrates.CK-M1 had been cloned and sequenced from the cDNA-RDA pool of Japanese flounder during metamorphosis.It shows conserved sequences with other vertebrate and high similarity and identity with other teleost CK-M.CK-M1 expresses in various tissues of adult flounder,including skeletal muscle,purtenance and gill. Whole mount hybridization and Real-time PCR showed that the CK-M1 levels changed in a stage-specific manner during larval development and metamorphosis, with increasing at first and reach the climax at the beginning of metamorphosis,then decreasing as the morphological changes going,finally come back to the pre-metamorphosis level,suggesting the CK-M1 is one of the important part of development of flounder larvae.
Trypsinogen 2 precursor(TR2P) is the precursor of a major proteolytic enzyme precursor produced by pancreatic acinar cells.TR2P was selected by RDA between sinistral and dextral hybrid between Japanese flounder and stone flounder.It had been cloned and sequenced in Japanese flounder and stone flounder.Both of them showed high similarity and identity with other teleost TRP.TR2P expresses expecially in intestine.Real-time PCR showed that the TR2P levels increasd sharply at the beginning of Japanese flounder's metamorphosis,and kept higher expression than before till after metamorphosis,suggesting the TR2P is related with development of flounder larvae.Real-time PCR verified that the TR2P isolated in dextral pool showed more relativity to the dextral stone flounders than sinistral Japanese flounder, suggesting that TR2P's expression is also related to the genes that direct flatfishes' eye-position.
左偏的牙鲆与右偏石鲽的杂交子代基于相同的遗传背景且在变态时眼睛向不同方向偏转,成为了研究鲆鲽鱼类变态和眼睛偏转方向的难得的实验材料。本实验运用了差异显示技术(DDRT-PCR)和代表性差异杂交(RDA)两种基因筛选方法用来筛选变态前期的左偏和右偏杂交种中差异表达的基因,分别筛选出23和47个片段。实验结果显示RDA是基因筛选的可行方法之一。大部分的片段与眼睛偏转方向都没有直接关系,但是RDA筛选出的肌肉型肌酸激酶1(CK-M1)和胰蛋白酶原前体2(TR2P)经检验与鲆鲽鱼类的变态有关。
肌肉型肌酸激酶1作为催化能量产生的重要催化剂之一,从变态前与变态前期的牙鲆仔鱼的RDA实验中筛选出经克隆测序,得到全序列。其序列与其他鱼类和哺乳动物的CK-M1的相似性很高。在牙鲆成鱼中,CK-M1在各个组织中都有较高的表达,特别是肌肉,内脏和鳃中。整体原位杂交和实时定量PCR结果显示CK-M1在变态期间的牙鲆仔鱼中的表达情况与变态紧密相关,随着变态的开始增加到高峰,之后下降,变态结束后,其表达量也下降到与变态前一样的水平。这些可以说明,CK-M1与牙鲆仔鱼的变态过程有着密切的关系。
胰蛋白酶原前体2是动物消化道中由胰腺细胞分泌的重要的消化酶之一。从牙鲆与石鲽杂交种的RDA实验中筛选出经分别在牙鲆和石鲽中克隆测序,分别得到全序列。其序列与其他鱼类的TR2P的相似性较高。在牙鲆成鱼中,TR2P在各个组织中都有表达,在肠道中的表达量最高。实时定量PCR结果显示TR2P在变态期间的牙鲆仔鱼中的表达情况与变态紧密相关,随着变态的开始突增,之后稍有下降,但一直维持高于变态前仔鱼的表达量直到变态完毕。可以说明,TR2P与牙鲆仔鱼的变态过程有着密切的关系。而且,RDA右偏库中筛选出的TR2P片段与同是右偏的父本石鲽的TR2P之间相似性高于与母本牙鲆,猜测其与鲆鲽鱼类的眼睛偏转方向相关。
Vertebral asymmetry in flatfish is the most significant among vertebrates,with both eyes being located on only one side(the ocular side) of the body.Changes in body proportion occur during flatfish metamorphosis,which exhibit extensive morphological and functional changes in various organs.There is little molecular reason to help understand the mechanism of flounders' eye migration,but still need to be proved.
The hybrids between Japanese flounder and stone flounder showed mixed eye-location:with both dextral type and sinistral type,and thus become good samples for studying the eye-migration problems,mRNAs from 22dph sinistral and dextral hybrids larvae were screened with the classical differential display RT-PCR (DD-RT-PCR) and representational difference analysis of cDNA(cDNA-RDA),and 23 and 47 putative fragments were isolated respectively.Most of the fragments isolated were not involved into eye migration specifically.But cDNA fragments of creatine kinase and trypsinogen 2 precursor genes isolated by cDNA-RDA exhibited stage specific expression patterns,indicating that they were related with energy supply and physiological changes during metamorphosis of flatfish.The present results showed that cDNA-RDA is one of the capable methods in isolating differentially expressed genes.
Creatine kinase(CK) catalyzes the reversible reaction of phosphorylation of creatine by ATP,which produces phosphorycreatine,a high-energy phosphagen in all vertebrates.CK-M1 had been cloned and sequenced from the cDNA-RDA pool of Japanese flounder during metamorphosis.It shows conserved sequences with other vertebrate and high similarity and identity with other teleost CK-M.CK-M1 expresses in various tissues of adult flounder,including skeletal muscle,purtenance and gill. Whole mount hybridization and Real-time PCR showed that the CK-M1 levels changed in a stage-specific manner during larval development and metamorphosis, with increasing at first and reach the climax at the beginning of metamorphosis,then decreasing as the morphological changes going,finally come back to the pre-metamorphosis level,suggesting the CK-M1 is one of the important part of development of flounder larvae.
Trypsinogen 2 precursor(TR2P) is the precursor of a major proteolytic enzyme precursor produced by pancreatic acinar cells.TR2P was selected by RDA between sinistral and dextral hybrid between Japanese flounder and stone flounder.It had been cloned and sequenced in Japanese flounder and stone flounder.Both of them showed high similarity and identity with other teleost TRP.TR2P expresses expecially in intestine.Real-time PCR showed that the TR2P levels increasd sharply at the beginning of Japanese flounder's metamorphosis,and kept higher expression than before till after metamorphosis,suggesting the TR2P is related with development of flounder larvae.Real-time PCR verified that the TR2P isolated in dextral pool showed more relativity to the dextral stone flounders than sinistral Japanese flounder, suggesting that TR2P's expression is also related to the genes that direct flatfishes' eye-position.
引文
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77. Williams S R (1901). The changes in the facial cartilaginous skeleton of the flatfishes, Pseudopleuronectes americanus (a dextral fish) and Bothus maculates (sinistral). Science New Ser 13, 378-379.
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80.Yamano,K.and Miwa,S.(1998).Differential gene expression of thyroid hormone receptor alpha and beta in fish development.Gen.Comp Endocrinol.109,75-85.
81.Yamano,K.,Miwa,S.,Obinata,T.,and Inui,Y.(1991).Thyroid hormone regulates developmental changes in muscle during flounder metamorphosis.Gen.Comp Endocrinol.81,464-472.
82.Yamano,K.,Takano-Ohmuro,H.,Obinata,T.,and Inui,Y.(1994b).Effect of thyroid hormone on developmental transition of myosin light chains during flounder metamorphosis.Gen.Comp Endocrinol.93,321-326.
83.Yeh C H,,P.J.H.S.,Wu Y C,and et al(2004).Differential2display polymerase chain identifies nicotinamide adenine dinucleotide ubiquone oxidoreductase as an ischemia/ reperfusion regulated gene in cardiomyocytes.Chest 124,1386-1390.
84.陈阅增,张宗炳,冯午,等(1997),普通生物学,高等教育出版社
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89.鲍宝龙,张臻字,龚小玲,等.(1999).外源甲状腺激素及可的松对牙鲆早期发育阶段生长、发育和变态的影响[J].上海水产大学学报,8(3):242-245.
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81.Yamano,K.,Miwa,S.,Obinata,T.,and Inui,Y.(1991).Thyroid hormone regulates developmental changes in muscle during flounder metamorphosis.Gen.Comp Endocrinol.81,464-472.
82.Yamano,K.,Takano-Ohmuro,H.,Obinata,T.,and Inui,Y.(1994b).Effect of thyroid hormone on developmental transition of myosin light chains during flounder metamorphosis.Gen.Comp Endocrinol.93,321-326.
83.Yeh C H,,P.J.H.S.,Wu Y C,and et al(2004).Differential2display polymerase chain identifies nicotinamide adenine dinucleotide ubiquone oxidoreductase as an ischemia/ reperfusion regulated gene in cardiomyocytes.Chest 124,1386-1390.
84.陈阅增,张宗炳,冯午,等(1997),普通生物学,高等教育出版社
85.张孝威,何桂芬,沙学坤.(1965)牙鲆和条鳎卵子及仔稚鱼的形态观察[J].海洋与湖沼,7(2):158-17
86.李思忠,王惠民.中国动物志[M].北京:科学出版社,1995.14.
87.刘立明(1996).不同温度条件下牙鲆变态期生长发育变化的研究[J1.海洋科学,4:58-63.
88.张臻字.鲍宝龙.(1999),鱼类早期发育阶段甲状腺激素的作用[J].上海水产大学学报,8(1):68-7
89.鲍宝龙,张臻字,龚小玲,等.(1999).外源甲状腺激素及可的松对牙鲆早期发育阶段生长、发育和变态的影响[J].上海水产大学学报,8(3):242-245.
90.#12